In microscopy for life sciences it is desirable to scan wide areas at high resolution and practical cost.
In the case of histology it is desirable to scan and store wide area views of microscope slides that carry tissue samples, cell cultures, arrays of diagnostic reagents exposed to blood, etc. Typical microscope slides have a viewable area of 2.5 cm by 7.5 cm.
In biotechnology research it is desirable to perform wide field of view microscopy of tissue cultures, DNA arrays on slides, DNA chips, segregated samples from gel electrophoresis, etc.
Such scanning is done to form images of objects, to read fluorescent emissions, or to illuminate, measure, alter or otherwise treat wide areas or discrete locations distributed over wide areas.
It is usually desirable to perform the microscopy tasks at as high a speed as possible, consistent with the amount of light that is received from the object.
Combining wide field microscopy with high speed can achieve telepathology via phone lines and satellite, more efficient evaluation of hybridization arrays, optical mapping of enzymatic restriction maps, etc.
A particularly important potential for high speed, wide field of view microscopy is the Human Genome project. By the year 2005 it is the goal to sequence the entire human genome of some 3 billion nucleotides, of which only some few million have been sequenced in the first five years of the project. The principles articulated here provide a way to speed this research.
High speed, wide area scanning, made available in a low cost system, can contribute to efficiencies in capital equipment and manpower not only in life sciences and biotechnology research, but also in the semiconductor industry and other technologies where microscopy is applicable.
According to one of the present contributions, it has been realized that wide field of view microscopy as well as high speed microscopy can be practically achieved by incorporating a micro objective lens in a limited rotation scanning structure of low moment of inertia. Where X, Y raster formats are desired the detected data from scan arcs is interpolated to equally spaced data points in an X, Y raster format. By this system images can be formed, manipulated, zoomed upon and analyzed from both macro and microscopic perspectives in a highly efficient manner. xe2x80x9cMicro lensxe2x80x9d as used here refers to lens assemblies weighing less than 2 grams and includes a single lens element having weight that is significantly less than 1 gram. xe2x80x9cMicro objectivexe2x80x9d as used here refers to movable micro lenses which either constitute the objective or constitute the first part of a multi element objective in which other elements are stationary.
Rather counter-intuitively, in particular, it is found that, a xe2x80x9cfirst in its classxe2x80x9d wide field of view microscope, or high speed microscope, can be achieved by employing a limited rotation driver, or galvanometer, carrying an extremely small field of view aspheric scanning micro objective lens.
Micro lenses, and in particular, aspheric micro lenses have a basic feature by which they differ from conventional microscope objectives. They are capable of focusing only on a very small spot, whereas high magnification commercial microscope objectives that use limited rotation techniques typically focus on an area greater than 100 microns in diameter.
While it is possible for the rotationally oscillating structure that carries the micro lens to also carry its own light source, in presently preferred cases the light source and detector assemblies remain stationary and communicate with the rotating objective by a periscope assembly that involves a pair of reflectors on the rotating, low moment of inertia structure.
The rotary micro lenses can avoid aberration effects by operating on-axis, and by the use of a single or a very few micro optical lens elements in the rotating assembly. Chromatic aberration is avoided in use of such micro lenses by manipulation of the different wavelengths in the stationary portion of the optical path. The light rays of various wavelengths are brought to focus at different points in the optical path in a compensating relationship that is predetermined to offset the chromatic aberration characteristic of the micro lens.
In applications of the new techniques to fluorescence microscopy, a micro objective lens mounted for limited rotation scanning, and having a large numerical aperture, is effective to collect the low intensity fluorescing wavelength in a cost-effective manner.
While various types of illumination may be employed with microscopes employing principles that have been discussed, it is advantageous to employ laser illumination for transmission and reflectance microscopy and in fluorescence readers. The new techniques are useful with advantage in some instances with a single color laser. With multiple lasers producing two colors, or three or more colors, it is possible to make multiple passes over the object, e.g. one for each color. Advantageously, however, examination of all colors is performed simultaneously, to conduct the entire chromatic examination in one pass.
The basic idea, to employ a tiny, low mass lens, preferably an aspheric lens, mounted in a low moment of inertia, limited rotation assembly for on-axis rotational scanning structure, can employ lenses made in a number of different ways. While a commercial glass lens made for fiber optic communications by a gel molding technique developed by Corning has been proposed, for present practical cases, the rotating lens may be molded of acrylic or styrene resin using well known lens design and molding techniques and grinding, machining and polishing techniques, etc.
For many systems of interest, an effective field of view for the limited rotation scanning microscope system is at least 1 cm square and preferably 1 inch (2.5 cm) by 3 inches (7.5 cm) or, for large microscope slides or the like, 3 inches by 4 inches, or more.
Resolution to fit the microscopic need can be readily achieved. In some dermatology applications, for instance, one may be interested to view cells which are 5 or 50 microns in dimension. In this case a micro lens with one micron resolution or greater is desirable.
For most practical applications of combined wide field of view and high speed limited rotation scanning, where there is an abundance of detected radiation, the numerical aperture (NA) of the scanning objective lens element is no less than about 0.5.
In fluorescence applications, the detected light levels are lower and the considerations are different from those of imaging. NA values in excess of 0.6, and as high as the order of 0.7 or 0.8 and even 0.9, near the theoretical limit in air, are obtainable and of significant advantage. The illumination spot size in fluorescence detection is often relatively large in the preferred embodiments, between 1 and 15 microns, and the energy collection ability of the lens, related to numerical aperture, is important. A limited rotation aspheric lens with NA of 0.8 enjoys a benefit of about 3 in light collection over a lens of 0.5 numerical aperture. Thus while employing large illumination spot sizes in limited rotation fluorescence microscopy, the aspheric micro lenses with high numerical aperture are of considerable advantage in low cost, relatively high speed applications.
Another contribution presented here is the use, with the rotary scanning structure, of a stationary periscope that extends closely over the object to conduct light from the stationary source to a stationary mirror directed along the axis of rotation to a reflector on the rotary assembly, thence to the rotating objective lens.
For many applications it is advantageous to move the object continuously under the limited rotation scanning head. Another contribution presented here concerns the reduction of scan overlap inefficiencies in such a system by introducing compensating motions of the beam relative to the rotating lens. When an objective lens is oscillated in a circular arc and the object is relatively translated continuously underneath (by translation of the object or translation of the axis of the rotating structure), a generally curved triangular scan pattern occurs upon the object and the object is not scanned uniformly when the image is acquired in both cw and ccw (back and forth) scans. If, for instance, the uniform spot size is such that the spot in the center of a scan arc, aligned with the direction of the translation, touches the path of the spot in the next successive scan, the scans will overlap considerably toward the apices of the curved triangular shaped wave pattern, while in the divergent regions of the pattern, areas of the object will be missed by the scan arcs. A compensating motion is introduced to the light path in the rotary system to cause successive scan paths to have a substantially uniform spacing over their useful length. This is implemented by moving the beam radially relative to the objective lens as rotation of the objective lens occurs, the objective lens being selected to have field of view of a minimum dimension of twice or more the spot size, so that the beam remains on the lens throughout the compensating excursion. For instance for a 5 micron diameter focused spot size, an objective lens having a minimum field of view of 10 microns, plus any amount necessary to facilitate alignment, is employed.
A simple device for achieving the compensatory motion is a dithering folding mirror located in the stationary optical path that addresses the rotating structure. A piezo-electric crystal dithers the mirror in synchronization with the rotary oscillations of the arm. This causes the beam to oscillate radially on the lens as the arm rotates. Instead of a piezo electric dither mirror, other reflecting devices may be dithered, or the compensating motion may be introduced into the beam path by other means, for instance by acousto-optic deflectors, electro-optic deflectors, rotary cranks or other moving linkages driven by motors.
The dither feature may be employed in various other ways, for instance to enable multiple scans of the object while the object is stationary relative to the scan arm axis.
Another contribution presented here is an efficient interpolation scheme and algorithm that converts scanned arc data values along arcuate scan lines to the uniformly spaced points of a raster format.
The specific construction details of the presently preferred implementations are also unique, and constitute contributions to microscope technology. While low mass mirrors are preferred in the moving system it will be appreciated that other reflectors, such as prisms, may be employed and other mechanical and electronic systems can be employed using concepts presented here.
Besides biological and life science applications, certain of the concepts have application to the silicon device industry, e.g. to inspect the relationship of features of an electronic device, such as inspection for co-planarity of features on a semiconductor chip such as a ball grid array used for making electrical connections to the chip.
By combining a confocal assembly with the limited rotation microscope described, the very shallow depth of field achieved enables verification that all legs of a silicon device are co-planar, while all of the data for the entire chip is captured rapidly in one bite (one wide view scan sequence). Likewise, one may perform three-dimensional mapping of features of silicon devices, living cells, or other objects.
According to one of the contributions, in a wide field of view, limited rotation scanning microscope for examination of a surface of an object, a scanning assembly is provided which comprises an oscillating rotary support structure associated with a driver and constructed to travel in periodic motion over the object to be viewed in a predetermined arcuate scan path over a scan range of at least 1 mm, a micro objective lens mounted on the rotary oscillating support structure, the micro objective lens characterized in having weight of less than about 2 grams, the lens mounted on the support structure with its axis normal to the surface of the object for essentially on-axis scanning throughout the arcuate scan range, and the driver for the support structure adapted to oscillate the support structure to cause on-axis scanning of the object.
Preferred embodiments of this aspect have one or more of the following features.
A reflecting system is mounted on the rotary support structure to define a light path communicating with the micro lens along the axis of the lens, the reflecting system constructed to maintain this optical path in optical communication with a stationary optical system over a light path of fixed length throughout the range of travel of the rotary oscillating support structure.
The micro objective lens is an aspheric lens.
The micro objective lens forms the entire objective of the microscope or it cooperates with stationary optical elements to form the objective of the microscope.
The oscillating assembly has a moment of inertia less than 25 gm-cm2.
Stationary optics produce at least two beams of different wave lengths and a merging system is constructed to merge the beams into a single illuminating beam directed to the micro objective lens. Preferably, where the micro objective lens has characteristic chromatic aberration, at least one device is included in the path of at least one of the beams to cause rays of one wave length to focus at a point different from the point of focus of rays of another wave length, the different focusing characteristics of the rays being redetermined in relation to the chromatic aberration characteristic of the objective lens to enable focus of the respective wave lengths, by the objective lens, upon the same point on the object.
Another of the contributions is a wide field of view limited rotation scanning microscope system which comprises the rotating, micro lens assembly described, combined with a translation system for producing relative linear movement over a translation range of an object to be scanned relative to the rotary support structure, the direction of translation being substantially normal to the center region of the limited rotation scan path.
Preferred embodiments of this aspect have one or more of the following features:
The microscope system is constructed and arranged to record an image area of at least one square centimeter of the surface being examined, the numerical aperture of the lens, its field of view, the scan range and the translation range being cooperatively selected to produce, for a given wave length, at least one million picture elements per cm2 of area scanned.
The wide field of view scanning microscope is constructed to produce images in a transmission or reflection mode, the numerical aperture of the micro objective lens being at least about 0.5.
Preferably the field of view of the micro objective lens is less than about 25 microns, and in many cases is less than about 10 microns.
The wide field of view scanning microscope is constructed to detect fluorescence stimulated by a spot of light passing through the micro objective lens, in which the numerical aperture of the scanning objective lens is greater than 0.6, preferably the field of view of the micro objective lens being less than about 25 microns.
The microscope system is constructed as a transmission microscope, the stationary optics including at least one stationary light source arranged to launch light to the micro objective lens to illuminate a spot on the object being viewed, and a detector system is disposed on the opposite side of the object being viewed.
The microscope system is constructed as a reflectance microscope, the stationary optics including at least one stationary light source arranged to launch light to the micro objective lens to illuminate a spot on the object being viewed, and a detector system is arranged to receive, via the micro objective lens, light reflected from the region being illuminated by the objective lens.
The microscope system is constructed to perform as a fluorescence reader, the stationary optics including at least one stationary light source arranged to launch light to the rotating micro objective lens to illuminate a spot on the object being read with a wave length predetermined to excite a fluorophor possibly present in the object, and a detector is arranged to receive, via the micro objective lens, fluorescing light from the fluorophor at a different wavelength emitted from the region being illuminated by the micro objective lens.
The microscope system is constructed to image detected light upon a pin hole preceding a detector to serve as a confocal microscope. Preferably the objective lens has a numerical aperture greater than 0.6 in this arrangement.
The microscope as a fluorescent reader is constructed and arranged so that the micro objective lens projects, on to the object, an illuminating spot between about 1 to 50 microns in diameter, the micro objective lens having a numerical aperture of about 0.6 or more for collection of relatively low intensity fluorescing radiation.
The scanning microscope has its axis of rotation of the rotary support structure stationary and the translation system for producing relative linear movement comprises a linear stage constructed to move the object to be viewed under the oscillating rotary structure.
The scanning microscope has stationary optics which include a reflector disposed on the axis of rotation of the rotary structure, and a reflector on the rotary structure is disposed on the axis of rotation, the two reflectors arranged in an optical path between the stationary optics and the scanning objective lens throughout the range of rotation of the oscillating rotary structure. Preferably the stationary optics includes a detector to detect light collected by the rotating micro objective lens from the object being scanned.
Preferably stationary optics include at least one stationary light source arranged to launch light to the objective lens to illuminate a spot on the object being viewed.
The scanning microscope system has stationary optics which include a path-deflecting device arranged to vary the portion of the micro objective lens lying in the optical path. In certain preferred embodiments, this feature is combined with a translation system in the manner that the path-deflecting device adjusts the relationship of successive scan paths upon the object being scanned. Preferably in systems in which the object is scanned during both clockwise and counterclockwise rotation of the oscillating rotary support structure, the adjustment made is in the sense of making more uniform, along the length of the scan path, the spacing between the mid lines of the successive scan paths.
The path-deflecting device for a microscope system is a dithered reflector driven in synchronism with the rotary oscillating support structure, preferably this device being a dithered mirror.
The path-deflecting device is an acousto-optical or electro-optical deflector driven in synchronism with the rotary oscillating support structure.
The wide field of view scanning microscope includes a position detector for detecting the position of the oscillating assembly, and including a data collection system that collects data at selected positions determined by the position detector. Preferably a control system for the driver includes a servo control loop that includes the position detector. Also, preferably the position detector is associated directly with the oscillating support structure to determine its position directly; preferably, also, the driver is an electric motor controlled by a servo control loop controlled by the directly determined position of the oscillating rotary support structure.
The wide field of view scanning microscope has the micro objective lens spaced from the center of rotation of the support structure more than 1 cm, the moment of inertia of the rotary structure, excluding the armature of the driver, is of the order of about 25 gm-cm2, for example 30 gm-cm2. Preferably, when combined with a translation system, the frequency of oscillation of the rotary oscillating structure produces in excess of about 10 scan line acquisitions per second. Preferably the radial distance is about 2.5 cm or greater.
The scanning microscope is in the form of a transmission or reflection microscope in which the driver for the rotary oscillating structure oscillates at a frequency of the order of 50 Hz or higher.
The wide field of view scanning microscope has a data collection control system which times the data collection during the scan motion to align data collection points with rows of a predetermined rectilinear raster grid. Preferably the data system converts the data to the raster grid by averaging for each point on the grid, the value of each of two data points in the raster row on either side of the grid point, the values weighted by their respective distances from the grid point in question.
According to another of the contributions, a limited rotation scanning microscope for examination of an object comprises in combination, an aspheric micro objective lens which serves either as the entire objective of the system or as the movable element of a multi element objective lens, having a field of view less than about 20 microns and a numerical aperture greater than about 0.5, a lens-carrying arm mounted and driven to rotate in an arc, in oscillating motion, about an axis that lies normal to the general plane of the object to be examined, the micro objective lens being mounted on the arm at a position spaced from the axis of rotation of the arm so that the micro objective lens is swept in an arc by rotation of the arm, the axis of the micro objective lens being normal to the plane of the surface to be examined, the axis of rotation being stationary, a translating mechanism being arranged to translate the surface to be examined under the rotating micro objective lens, and a light source mounted on a stationary support and associated with optical elements defining an optical path for light to pass from the light source to the micro objective lens, thence to a spot on the surface to be examined.
Preferred embodiments of this aspect have one or more of the following features.
The scanning microscope includes a light source mounted on a stationary support and associated with optical elements defining an optical path for light to pass from the light source to the micro objective lens, thence to a spot on the surface to be examined.
The scanning microscope is in the form of a transmission microscope, light from a spot passes through the micro objective lens and object reaches a detector.
In other forms of the scanning microscope, light from a spot of light passed through the micro objective lens and to the object, returns through the micro objective lens to a detector. In certain cases such a scanning microscope is constructed to read fluorescing light from the object.
The scanning microscope includes a control system for producing coordinated rotation and translation of the object, the microscope constructed to receive data from scan paths generated during clockwise and counterclockwise rotation of the arm, the control system including a compensatory system that varies the relationship between movement of the micro objective lens and translation of the object in a manner tending to make substantially uniform the distance between the mid-lines of the successive scan paths. Preferably, the compensatory system varies the position on the micro objective lens of the light path communicating with the stationary light source, preferably the compensating system comprising a dither mirror.
In any of the microscope systems previously described above that employs a table to receive the object, the table is preferably associated with three adjustable elevators to raise, lower and tilt the table for focusing, and a control system is constructed to conduct a prescan of the object in which data concerning orientation is stored, and a control system responsive to the stored data is effective to actuate the elevators as scanning proceeds to maintain the object in focus.
In microscopes systems previously described above where the micro objective lens is the movable portion of a multi element objective lens and other optical elements are stationary, all optical elements cooperate to perform in a manner similar to a conventional multi element objective lens. The stationary optical elements can cooperate with the movable micro objective lens to optimize energy collection and transfer to the light sensor.
Another contribution comprises, in general, a dither mirror construction comprising a mirror mounted on a flexure and a piezo crystal associated with the mirror in the manner to cause deflection of the mirror on its flexure. This dither mirror is preferably employed in the various scanning microscopes and methods described.
Another contribution comprises, in general, a method of scanning an object in manner to form an image comprising moving in scanning motion a lens of mass less than about 2 gm on a moving structure, directly detecting the position of the lens while collecting light from the object with the lens, and compiling detected data based on positions directly detected at the time of taking the data.
Another contribution comprises, in general, a method of scanning an object employing rotating a lens on an arm in scanning arcs over an object that is translating relative to the axis including deflecting the optical path relative to the lens in a compensatory motion in the sense tending to make substantially uniform the spacing between adjacent scan lines upon an advancing object.
Another contribution comprises, in general, a scanning microscope comprising a micro objective lens mounted to move in scanning motion over an object, stationary optics that produces at least two beams of different wave lengths and a merging system constructed to merge the beams into a single illuminating beam directed to the micro objective lens, the micro objective lens having characteristic chromatic aberration, and a device is included in the path of at least one of the beams to cause rays of one wave length to focus at a point different from the point of focus of another wavelength, the different focusing characteristics of the wavelengths being predetermined in relation to the chromatic characteristic of the micro objective lens to enable focus of the respective wave lengths, by the micro objective lens, upon the object.
In certain preferred embodiments stationary optical elements cooperates with the micro objective lens mounted on the rotatably oscillating arm to form an effective objective of the system.
Another contribution comprises, in general, a rotary scanning system producing arcuate scan motion having a data collection control arranged to time the data collection during the arcuate scan motion to align data collection points with rows of a predetermined raster grid to which the data is to be converted. Preferably, this system includes a data conversion system arranged to convert data to the raster grid by averaging for each raster point the value of each of the two data points in the row on either side of the raster point, the values weighted by their respective distances from the raster point in question.
Other features of the invention will be understood from the following description of preferred embodiments.